1. Field of the Invention
The present invention relates generally to the fields of vector biology and gene therapy. More specifically, the present invention relates to the production of recombinant adenoviral vectors with replacement of fibers for cell-specific targeting with concomitant elimination of endogenous tropism.
2. Description of the Related Art
Adenovirus interacts with eukaryotic cells by virtue of specific receptor recognition of domains in the knob portion of the fiber protein (21-23) which protrude from each of the twelve vertices of the icosahedral capsid. Recombinant adenovirus vectors are used in a number of gene therapy applications (21, 35, 38). This fact has derived principally from the high levels of gene transfer achievable with this vector approach both in vitro and in vivo.
Recombinant adenovirus vectors are distinguished from other systems by their unique ability to accomplish in situ gene delivery to differentiated target cells in a variety of organ contexts (5, 6, 9, 10, 12, 20, 25, 27, 29, 31). This has allowed the utilization of recombinant adenoviral vectors as an approach to treat inherited genetic diseases, such as cystic fibrosis, whereby the delivered vector may be contained within the target organ (4-13). In addition, the ability of the adenoviral vector to accomplish in situ tumor transduction has allowed the development of a variety of anti-cancer gene therapy approaches for loco-regional disease (14-18).
Adenoviral vectors can accomplish in vivo gene delivery to a variety of organs after intravenous injection. In these instances, gene transfer frequencies have been sufficiently high to correct inherited metabolic abnormalities in various murine models. Thus, adenoviral vectors fulfill two requirements of an intravenously administered vector for gene therapy: systemic stability and the ability to accomplish long-term gene expression following high efficiency transduction of muscle cells.
Adenoviruses suffer, however, from the disadvantage that the widespread distribution of the adenovirus cellular receptor precludes the targeting of specific cell types. This lack of tropism of adenoviral vectors would result in a decrease in the efficiency of transduction, as the number of virus particles available for delivery to the target cells would be decreased by sequestration by nontarget cells. Furthermore, this would allow ectopic expression of the delivered gene, with unknown and possibly deleterious consequences. Therefore, a means must be developed to redirect the tropism of the adenovirus vector specifically to target cells and permit gene delivery only to affected organs.
To this end, several groups have reported genetic modifications to the knob domain of adenovirus fiber protein and incorporation of such chimeric fibers into virion. For instance, Stevenson et al. (35) and others (24) reported successful generation of Ad5 virions containing fibers consisting of the tail and shaft domains of Ad5 fiber and the knob domain of Ad3, respectively. In addition, Michael et al. (30) demonstrated the incorporation of the gastrin-releasing peptide into the carboxy terminus of recombinant Ad5 fiber. This finding was extended by Legrand et al. (30a) who achieved rescue of recombinant adenovirus vectors containing such fibers. Wickham et al. (41) described the generation of recombinant virus containing fibers with carboxy-terminal polylysine sequences. These studies have established key feasibility issues with respect to this genetic approach but have also demonstrated a number of limiting factors, including the size of the ligand, which may disrupt the correct folding of the fiber protein.
The prior art remains deficient in the lack of effective means to produce recombinant adenoviral vectors with combination of novel targeting and ablation of native tropism. The present invention fulfills this longstanding need and desire in the art.